RESUMEN
Cultured mouse embryonic stem cells are a heterogeneous population with diverse differentiation potential. In particular, the subpopulation marked by Zscan4 expression has high stem cell potency and shares with 2 cell stage preimplantation embryos both genetic and epigenetic mechanisms that orchestrate zygotic genome activation. Although embryonic de novo genome activation is known to rely on metabolites, a more extensive metabolic characterization is missing. Here we analyze the Zscan4+ mouse stem cell metabolic phenotype associated with pluripotency maintenance and cell reprogramming. We show that Zscan4+ cells have an oxidative and adaptable metabolism, which, on one hand, fuels a high bioenergetic demand and, on the other hand, provides intermediate metabolites for epigenetic reprogramming. Our findings enhance our understanding of the metastable Zscan4+ stem cell state with potential applications in regenerative medicine.
Asunto(s)
Células Madre Embrionarias de Ratones , Factores de Transcripción , Animales , Blastocisto/metabolismo , Metaboloma , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Estrés Oxidativo , Factores de Transcripción/metabolismoRESUMEN
Y-box binding protein 1 (YBX-1 or YB-1) is an oncoprotein that promotes replicative immortality, tumor cell invasion and metastasis. The increase in the abundance of YB-1 in the cell or YB-1 translocation from the cytoplasm to the nucleus is characteristic of malignant cell growth. We have previously reported that ΔNp63α, a transcription factor that is known to play a pivotal role in keratinocyte proliferation and differentiation, promotes YB-1 nuclear accumulation. Here, we show that YB-1 is highly expressed in proliferating keratinocytes and is down-regulated during keratinocyte differentiation. ΔNp63α reduces YB-1 protein turnover and leads to accumulation of ubiquitin-conjugated YB-1 into the nucleus. Reduction of YB-1 protein level, following treatment with a DNA-damaging agent, is inhibited by ΔNp63α suggesting that YB-1 and ΔNp63α interplay can support keratinocyte proliferation and protect cells from apoptosis under genotoxic stress.
Asunto(s)
Queratinocitos/citología , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteína 1 de Unión a la Caja Y/química , Ciclo Celular , Diferenciación Celular , Línea Celular Tumoral , Regulación hacia Abajo , Humanos , Queratinocitos/metabolismo , Estabilidad Proteica , Proteína 1 de Unión a la Caja Y/metabolismoRESUMEN
Cutaneous squamous cell carcinomas (SCCs) typically lack somatic oncogene-activating mutations and most of them contain p53 mutations. However, the presence of p53 mutations in skin premalignant lesions suggests that these represent early events during tumor progression and additional alterations may be required for SCC development. SCC cells frequently express high levels of ΔNp63α and Y-box binding 1 (YB-1 or YBX1) oncoproteins. Here, we show that knockdown of YB-1 in spontaneously immortalized HaCaT and non-metastatic SCC011 cells led to a dramatic decrease of ΔNp63α, cell detachment and death. In highly metastatic SCC022 cells, instead, YB-1 silencing induces PI3K/AKT signaling hyperactivation which counteracts the effect of YB-1 depletion and promotes cell survival. In summary, our results unveil a functional cross-talk between YB-1, ΔNp63α and the PI3K/AKT pathway critically governing survival of squamous carcinoma cells.
Asunto(s)
Carcinoma de Células Escamosas/genética , Proteína Oncogénica v-akt/genética , Fosfatidilinositol 3-Quinasas/genética , Neoplasias Cutáneas/genética , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética , Proteína 1 de Unión a la Caja Y/genética , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Proliferación Celular/genética , Supervivencia Celular/genética , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Proteína Oncogénica v-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/genética , Neoplasias Cutáneas/patología , Proteína p53 Supresora de Tumor/genética , Proteína 1 de Unión a la Caja Y/biosíntesisRESUMEN
The Y-box binding protein 1 (YB-1) belongs to the cold-shock domain protein superfamily, one of the most evolutionarily conserved nucleic acid-binding proteins currently known. YB-1 performs a wide variety of cellular functions, including transcriptional and translational regulation, DNA repair, drug resistance, and stress responses to extracellular signals. Inasmuch as the level of YB-1 drastically increases in tumor cells, this protein is considered to be one of the most indicative markers of malignant tumors. Here, we present evidence that ΔNp63α, the predominant p63 protein isoform in squamous epithelia and YB-1, can physically interact. Into the nucleus, ΔNp63α and YB-1 cooperate in PI3KCA gene promoter activation. Moreover, ΔNp63α promotes YB-1 nuclear accumulation thereby reducing the amount of YB-1 bound to its target transcripts such as that encoding the SNAIL1 protein. Accordingly, ΔNp63α enforced expression was associated with a reduction of the level of SNAIL1, a potent inducer of epithelial to mesenchymal transition. Furthermore, ΔNp63α depletion causes morphological change and enhanced formation of actin stress fibers in squamous cancer cells. Mechanistic studies indicate that ΔNp63α affects cell movement and can reverse the increase of cell motility induced by YB-1 overexpression. These data thus suggest that ΔNp63α provides inhibitory signals for cell motility. Deficiency of ΔNp63α gene expression promotes cell mobilization, at least partially, through a YB-1-dependent mechanism.
Asunto(s)
Movimiento Celular , Núcleo Celular/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteína 1 de Unión a la Caja Y/metabolismo , Transporte Activo de Núcleo Celular/genética , Línea Celular Tumoral , Núcleo Celular/genética , Supervivencia Celular/genética , Humanos , Isoformas de Proteínas , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética , Proteína 1 de Unión a la Caja Y/genéticaRESUMEN
Congenital Dyserythropoietic Anemia type II is an autosomal recessive disorder characterized by unique abnormalities in the differentiation of cells of the erythroid lineage. The vast majority of CDA II cases result from mutations in the SEC23B gene. To date, 53 different causative mutations have been reported in 86 unrelated cases (from the CDA II European Registry), 47 of them Italian. We have now identified SEC23B mutations in 23 additional patients, 17 Italians and 6 non-Italian Europeans. The relative allelic frequency of the mutations was then reassessed in a total of 64 Italian and 45 non-Italian unrelated patients. Two mutations, E109K and R14W, account for over one-half of the cases of CDA II in Italy. Whereas the relative frequency of E109K is similar in Italy and in the rest of Europe (and is also prevalent in Moroccan Jews), the relative frequency of R14W is significantly higher in Italy (26.3% vs. 10.7%). By haplotype analysis we demonstrated that both are founder mutations in the Italian population. By using the DMLE+ program our estimate for the age of the E109K mutation in Italian population is ≈2,200 years; whereas for the R14W mutation it is ≈3,000 years. We hypothesize that E109K may have originated in the Middle East and may have spread in the heyday of the Roman Empire. Instead, R14W may have originated in Southern Italy. The relatively high frequency of the R14W mutation may account for the known increased prevalence of CDA II in Italy.
Asunto(s)
Anemia Diseritropoyética Congénita/genética , Efecto Fundador , Frecuencia de los Genes , Mutación , Proteínas de Transporte Vesicular/genética , Sustitución de Aminoácidos , Anemia Diseritropoyética Congénita/sangre , Anemia Diseritropoyética Congénita/epidemiología , Análisis Mutacional de ADN , Europa (Continente)/epidemiología , Evolución Molecular , Femenino , Estudios de Asociación Genética , Haplotipos , Humanos , Italia/epidemiología , Masculino , Región Mediterránea/epidemiología , Polimorfismo de Nucleótido Simple , Prevalencia , Sistema de RegistrosRESUMEN
The prototype cold-shock Y-box binding protein 1 (YB-1) is a multifunctional protein that regulates a variety of fundamental biological processes including cell proliferation and migration, DNA damage, matrix protein synthesis and chemotaxis. The plethora of functions assigned to YB-1 is strictly dependent on its subcellular localization. In resting cells, YB-1 localizes to cytoplasm where it is a component of messenger ribonucleoprotein particles. Under stress conditions, YB-1 contributes to the formation of stress granules (SGs), cytoplasmic foci where untranslated messenger RNAs (mRNAs) are sorted or processed for reinitiation, degradation, or packaging into ribonucleoprotein particles (mRNPs). Following DNA damage, YB-1 translocates to the nucleus and participates in DNA repair thereby enhancing cell survival. Recent data show that YB-1 can also be secreted and YB-1-derived polypeptides are found in plasma of patients with sepsis and malignancies. Here we show that in response to oxidative insults, YB-1 assembly in SGs is associated with an enhancement of YB-1 protein secretion. An enriched fraction of extracellular YB-1 (exYB-1) significantly inhibited proliferation of receiving cells and such inhibition was associated to a G2/M cell cycle arrest, induction of p21WAF and reduction of Np63 protein level. All together, these data show that acute oxidative stress causes sustained release of YB-1 as a paracrine/autocrine signal that stimulate cell cycle arrest.
RESUMEN
Uncaria tomentosa (Willd.) DC. (Rubiacee), also known as uña de gato, is a plant that grows wild in the upper Amazon region of Peru and has been widely used in folk medicine to treat several health conditions including cancer. We have produced an aqueous extract from Uncaria tomentosa (UT-ex) and analyzed its effects on squamous carcinoma cells and immortalized HaCaT keratinocytes. Squamous cell carcinoma (SCC) is an uncontrolled growth of abnormal cells arising in the skin's squamous layer of epidermis. When detected at an early stage, SCCs are almost curable, however, if left untreated, they can penetrate the underlying tissue and become disfiguring. We have evaluated cell proliferation, apoptosis and the level of reactive oxygen species following UT-ex treatment. UT-ex affected cell cycle progression and reduced cell viability in a dose and time-dependent manner. From a mechanistic point of view, this delay in cell growth coincided with the increase of reactive oxygen species (ROS). Furthermore, PARP1 cleavage was associated to the reduction of Y-box binding protein 1 (YB-1) 36kDa, a nuclear prosurvival factor involved in DNA damage repair. These data indicate that UT-ex-induced cell death can be ascribed, at least in part, to its ability both to induce oxidative DNA damage and antagonize the mechanism of DNA repair relying upon YB-1 activity. They also show that non metastatic SCCs are more susceptible to UT-ex treatment than untransformed keratinocytes supporting the use of UT-ex for the treatment of precancerous and early forms of squamous cell carcinomas. Preliminary chemical investigation of UT-ex revealed the presence of hydrophilic low-medium molecular weight metabolites with anticancer potential towards squamous carcinoma cells.
Asunto(s)
Antineoplásicos/farmacología , Uña de Gato , Extractos Vegetales/farmacología , Apoptosis/efectos de los fármacos , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/metabolismo , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Queratinocitos/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/metabolismoRESUMEN
Depending on their genetic background (p53(wt) versus p53(null)), carcinoma cells are more or less sensitive to drug-induced cell cycle arrest and/or apoptosis. Among the members of the p53 family, p63 is characterized by two N-terminal isoforms, TAp63 and ΔNp63. TAp63 isoform has p53-like functions, while ΔNp63 acts as a dominant negative inhibitor of p53. We have previously published that TAp63 is involved in poly(ADP-ribose)polymerase-1 (PARP-1) signaling of DNA damage deriving from DNA topoisomerase I (TOP I) inhibition in carcinoma cells. In the present study, we treated MCF7 breast carcinoma cells (p53(+)/ΔNp63(-)) or SCC022 (p53(-)/ΔNp63(+)) squamous carcinoma cells with the TOP I inhibitor topotecan (TPT) and the PJ34 PARP inhibitor, to compare their effects in the two different cell contexts. In MCF7 cells, we found that PJ34 addition reverts TPT-dependent PARP-1 auto-modification and triggers caspase-dependent PARP-1 proteolysis. Moreover, TPT as single agent stimulates p53(ser15) phosphorylation, p53 PARylation and occupancy of the p21WAF promoter by p53 resulting in an increase of p21WAF expression. Interestingly, PJ34 in combination with TPT enhances p53 occupancy at the BAX promoter and is associated with increased BAX protein level. In SCC022 cells, instead, TPT+PJ34 combined treatment reduces the level of the anti-apoptotic ΔNp63α protein without inducing apoptosis. Remarkably, in such cells, either exogenous p53 or TAp63 can rescue the apoptotic program in response to the treatment. All together our results suggest that in cancer cells PARP inhibitor(s) can operate in the choice between growth arrest and apoptosis by modulating p53 family-dependent signal.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Proteínas de la Membrana/fisiología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Inhibidores de Topoisomerasa I/farmacología , Proteína p53 Supresora de Tumor/fisiología , Secuencia de Bases , Supervivencia Celular/fisiología , Cartilla de ADN , Humanos , Células MCF-7 , Procesamiento Proteico-Postraduccional , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Poly(ADP-ribose)polymerase 1 (PARP-1) inhibitors are thought as breakthrough for cancer treatment in solid tumors such as breast cancer through their effects on PARP's enzymatic activity. Our previous findings showed that the hydrophilic PARP inhibitor PJ34 enhances the sensitivity of p53 proficient MCF7 breast carcinoma cells to topotecan, a DNA Topoisomerase I (TOP 1) inhibitor. In the present study, we combine the classical TOP 1 poison camptothecin or its water-soluble derivative topotecan with PJ34 to investigate the potentiation of chemotherapeutic efficiency in MCF7 (p53(WT)), MDA-MB231 (p53(mut)) breast carcinoma cells and SCC022 (p53(null)) squamous carcinoma cells. We show that, following TPT-PJ34 combined treatment, MCF7 cells exhibit apoptotic death while MDA-MB231 and SCC022 cells are more resistant to these agents. Specifically, in MCF7, (i) PJ34 in combination with TPT causes a G2/M cell cycle arrest followed by massive apoptosis; (ii) PJ34 addition reverts TPT-dependent PARP-1 automodification and triggers caspase-dependent PARP-1 proteolysis; (iii) TPT, used as a single agent, stimulates p53 expression while in combination with PJ34 increases p53, TAp63α and TAp63γ protein levels with a concomitant reduction of MDM2 protein. The identification of p63 proteins as new players involved in the cancer cell response to TPT-PJ34 is relevant for a better understanding of the PARP1-dependent signaling of DNA damage. Furthermore, our data indicate that, in response to TPT-PJ34 combined chemotherapy, a functional cooperation between p53 and TAp63 proteins may occur and be essential to trigger apoptotic cell death.